PROJECT SUMMARY Mitochondrial cytochrome c release catalyzed by the B-cell lymphoma 2 (Bcl-2) family members Bax and Bak is the committed step to apoptosis, yet the upstream mechanisms leading to the `cell suicide' decision are not fully understood. The voltage-dependent anion channel isoforms (particularly VDAC 1 and 2), known for decades as non-selective mitochondrial outer membrane pores for exchange of metabolites such as ATP and pyruvate between the mitochondria and cytoplasm, have recently been found to interact with the pro-apoptotic pore formers Bax and Bak, thus implicating VDACs as apoptosis regulators. The Bcl-2 homology 3 (BH3) ?-helix domain is the means by which all Bcl-2 family members interact, thus raising the question of how integral membrane ?-barrel VDACs could interact with Bax and Bak. This proposal tests the hypothesis that all VDAC isoforms (VDAC 1-3) have a functional N-terminal BH3 domain and are thus novel members of the Bcl-2 family capable of interacting with both pro- and anti-apoptotic members. The hypothesis is based on strong sequence conservation of the VDAC N- terminus with Bcl-2 family BH3 domains, as well as in silico docking analysis of the VDAC N-terminus to the Bax `hydrophobic groove' receptor site for BH3 domains. The first specific aim will evaluate recombinant Bax and Bcl-2 binding to synthetic N-terminal VDAC peptides, identify VDAC residues important for peptide binding, and assess the ability of the peptides to activate or sequester Bax in a liposome permeabilization assay. The second specific aim will determine if a Bax or Bcl-2 chimera having its BH3 domain replaced with the VDAC BH3-like domain, can restore apoptosis sensitivity to Bax/Bak double knock out mouse embryonic fibroblasts, or increase resistance to apoptosis in Bcl-2 knockout mouse embryonic fibroblasts, respectively. The third specific aim will determine if mammalian or Drosophila VDACs can regulate mammalian Bax in an N-terminal domain-dependent manner in Drosophila melanogaster that are either partially or completely deficient for endogenous VDAC. Completion of these aims will advance the field of cell death regulation by identifying VDACs as new members of the Bcl-2 family possessing functional BH3 domains capable of interacting with established pro- and anti-apoptotic members.